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Abstract

Exosomes have emerged as an important mediator of intercellular communication. They are present in extracellular milieu and therefore, easily accessible by neighboring or distant cells. They carry mRNA, microRNAs and proteins within their vesicles and once internalized by recipient cells; they can modulate multiple signaling pathways with pleiotropic effects from inducing antiviral state to disease progression. We have previously shown that hepatitis C virus (HCV) infected hepatocytes or hepatoma cells harboring genome-length replicon secrete exosomes in culture supernatants. These exosomes are taken up by hepatic stellate cells (HSC) and activate them to induce fibrosis during HCV infection. Here, we describe detailed protocols for exosomes isolation and uptake of BODIPY labeled exosomes by hepatic stellate cells.

Discard the supernatant and resuspend cell pellet in 5 ml of complete media and transfer to a T-25 cm2 culture flask.

Incubate cells at 37 °C in a 5% CO2 incubator.

Observe culture flasks routinely under inverted microscope using 10x objective and split cells one day before the experiment.Note: Next day after revival, observe the culture flask for floating cells. If floating cells are seen, replace flask with fresh 5 ml of complete media. Hepatoma cells should be split in 1:3 to 1:5 ratio in every 3-5 days to maintain the cell growth.

BODIPY labeling of exosomes

Seed 2 x 106 Huh7.5 cells of ~50% confluency in a 100 mm plate and incubate at 37 °C in 5% CO2 incubator overnight. Next day, infect cells with HCV at a multiplicity of infection (MOI) of 0.1. Allow virus to adsorb onto the cells for 8 h in a minimum volume (2 ml) of DMEM with 2% of FBS and antibiotics. Wash the virus-infected cells 3 times with PBS.Note: Media with reduced serum concentration helps in virus infection. PBS should be kept at room temperature before use.

Dilute 10 μl of 1 mM of BODIPY 493/503 solution (see Recipes) to 10 ml of culture media to make the final concentration of 1 μM, mix well and add onto the virus infected Huh7.5 or Rep2a-Rluc cells.

Incubate cells for 1 h at 37 °C in 5% CO2 incubator.

Wash the cells 3 times with PBS.

Add 10 ml of fresh DMEM media with 2% of exosome depleted sera (see Recipes) onto the cells and incubate for either 3 days or 24 h for HCV infected Huh7.5 or Rep2a-Rluc cells respectively.

After incubation, collect culture supernatant and proceed for exosomes isolation.

Exosome isolation by ultracentrifugation

Collect 10 ml of culture supernatant from HCV infected Huh7.5 or Rep2a-Rluc cells and centrifuge at 300 x g at 4 °C for 5 min. Without disturbing the cell pellet, carefully transfer the supernatant to a new ultra-clear centrifuge tube.

To get rid of possible cell debris, centrifuge the supernatant at 2,000 x g for 10 min at 4 °C, followed by 26,500 x g for 30 min at 4 °C using SW41 Ti rotor by ultracentrifugation. Transfer the supernatant to a new ultra-clear centrifuge tube.

Then, centrifuge the supernatant using SW41 Ti rotor at 110,000 x g for 90 min at 4 °C by ultracentrifugation to isolate the exosomes. Discard the supernatant.

Wash the exosome pellet 2 times with PBS. For this, resuspend pellet in 10 ml of PBS and centrifuge at 110,000 x g for 60 min at 4 °C. Discard the supernatant. Repeat this step again.

Resuspend the final pellet referred to as exosomes in PBS to 1/20 of the original volume of culture supernatant for analysis.

Check the size distribution of exosomes by dynamic light scattering (DLS) using a Zetasizer Nano (Malvern Instruments). Size distribution analysis of exosomes by DLS is shown in Figure 1. Exosome size is measured as diameter in nm (d. nm) and Polydispersity index (PDI) indicates size distribution of exosomes within the sample.

Next day, expose LX2 cells with BODIPY labeled exosomes for 3 h (short) or 24 h (long).Note: Exosomes should be diluted in serumfree media from 1:2 to 1:10 before adding onto LX2 cells. Do not add exosomes directly onto the cells. Since exosomes are resuspended in PBS, longer incubation of cells with exosomes will detach cells from chamber slides. To observe any changes at molecular level such as gene expression, exosomes should be incubated with recipient cells for a minimum of 24 h.

It is very hard to observe evenness of exosome labeling by microscopy. They are too small to be individually resolved. Generally, under microscope, BODIPY labeled exosomes tend to clump together. Exosomes size vary from 50-150 nm in diameter, however, high protein content in exosomes increases the size up to 200 nm in diameter.

Two commonly used method for exosome isolation worked on different principles. Ultracentrifugation involves differential centrifugation. Successive rounds of centrifugation are intended to pellet down sequentially apoptotic bodies, cell debris, shedding vesicles and then, the exosomes are isolated based on size and density. It is a timeconsuming process involves multiple steps and potentially lead to exosomal aggregation. Exoquick method involves polymer based precipitation technique. There is a chance of contamination of exosomes with microvesicles and apoptotic bodies, however, exosomes can be isolated from very small amount of sample by this method.

Data analysis

Each experiment has been repeated at least three times to verify reproducibility.

Recipes

BODIPY stock solution

Prepare 5 mM (1.25 mg/ml) stock solution in DMSO

Store the stock solution at -20 °C wrapped in aluminum foil

Protect it from light and make smaller aliquots to avoid repeated freeze-thaw

Prepare 1 mM working solution in DMSO at the time of experiment

Exosome depleted serum
Ultracentrifuge FBS at 110,000 x g at 4 °C for 16 h using SW41 Ti rotor (Beckman Coulter), collect the supernatant and then pass through a 0.22 µm filter
Store the filtered exosome depleted serum at 4 °C for further use

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